Recent progress in elucidating the pathogenesis of Alzheimer's disease (AD) has centered on the apparent role of the 40-42 residue amyloid beta- peptide (Abeta) as a unifying pathological feature of the genetically diverse forms of this complex disorder. For example, DNA mutations have been identified in several distinct genes (four of which have been discovered to date) associating with familial AD (FAD). In addition, others have shown that the proteolytic processing of beta-amyloid precursor protein (betaAPP) is altered by many of these mutations in a way that results in increased production of Abeta peptides, particularly of the highly hydrophobic (and thus amyloidogenic) 42-residue form of Abeta (Abeta/42). Elevated production of Abeta/42 by FAD-linked genetic mutations indicates that the proteolytic cleavage of betaAPP by gamma- secretase may be a key element in the formation of amyloidogenic form of Abeta. A full understanding of the molecular basis of the substrate specificity of gamma-secretase requires knowledge of how FAD-associated genetic mutations affect the processing of betaAPP in AD. However, none of these enzymes responsible for the proteolytic cleavage of betaAPP have yet been identified. The objective of this study is to investigate the molecular mechanisms of gamma-secretase in the processing of betaAPP, with three specific aims. (1) Characterizing the substrate specificity of gamma-secretase cleavage of betaAPP. (2) Investigating the effects of the association of the transmembrane domain of betaAPP with membrane on gamma- secretase cleavage. (3) Investigating the effects of the localization of betaAPP to different cellular compartment on the function of gamma- secretase. Site directed mutagenesis will be used to vary either the properties of amino acid residues around the gamma-secretase cleavage site or to vary the length of the transmembrane domain of betaAPP. The exact identities of secreted Abeta-related peptides will be determined be measuring their molecular basses using immunoprecipitation and mass spectrometry. These studies will reveal fine detains concerning the cleavage specificities of gamma-secretase towards betaAPP, leading to better strategies to identify the as yet undiscovered enzyme(s).